Assay for High-Throughput Identification of Therapeutic Compounds

ABSTRACT

A solid supported branched linker assay system, including an alpha compound and a beta compounds reversibly tethered to a solid support; a branched linker coupled to the solid support that tethers the alpha and beta compounds to the solid support; the branched linker having two cleavable linkers that are chemically distinct from one another, wherein a first chemically distinct linker tethers the β compound to the branched linker and a second chemically distinct linker tethers the α compound to the branched linker; and at least two means for cleaving the chemically distinct linkers, wherein a first cleavage means is configured to selectively cleave a first chemically distinct linker and a second cleavage means is configured to selectively cleave a second chemically distinct linker.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No.62/310,746 entitled “Peptoid Library Agar Diffusion Assay forHigh-Throughput Identification of Antimicrobial Compounds” and filed on20 Mar. 2016, the contents of which are incorporated by reference in itsentirety.

GOVERNMENT INTERESTS

This invention was made with government support under Grant No. R03AI112861 awarded by the National Institutes of Health. The governmenthas certain rights in the invention.

All patents, patent applications, and publications cited herein arehereby incorporated by reference in their entirety. The disclosures ofthese publications in their entireties are hereby incorporated byreference into this application in order to more fully describe thestate of the art as known to those skilled therein as of the date of theinvention described and claimed herein.

This patent disclosure contains material that is subject to copyrightprotection. The copyright owner has no objection to the facsimilereproduction by anyone of the patent document or the patent disclosureas it appears in the U.S. Patent and Trademark Office patent file orrecords, but otherwise reserves any and all copyright rights.

FIELD OF THE INVENTION

In various exemplary embodiments, the present invention comprises asolid supported branched linker system and high-throughput screeningmethod for the identification of therapeutic compounds.

BACKGROUND OF THE INVENTION

The increasing prevalence of multi-drug-resistant (MDR) bacterialinfections in the clinic necessitates methods to rapidly identify potentnew antimicrobial agents that are effective against MDR bacteria.Antimicrobial resistance (AMR) is considered by the World HealthOrganization (WHO) to be a major threat to global public health,resulting in significant detrimental effects on mortality rates andeconomic growth due to the growing cost of bacterial infectiontreatment. A recent study predicted that the by the year 2050, AMR willresult in 10 million premature deaths per year worldwide and roughly$100 trillion USD in lost economic output. Bacterial resistance is agrowing problem due to increasing and improper use of antibioticscombined with the ability of bacteria to readily transmit informationfrom one microbe to another. Common mechanisms of bacterial resistanceinclude drug efflux pumps and enzymes that break down commonantibiotics, such as β-lactamases and aminoglycosides. Although there isa prevalent AMR problem, relatively few bioavailable antimicrobialtherapeutics have been identified that are resistant to these enzymes.

Accordingly, what is needed is a process and system for high-throughputscreening and identification of new antimicrobial drugs and compounds,including, but not limited to, antimicrobial peptides.

SUMMARY OF THE INVENTION

In various exemplary embodiments, the present invention comprises asolid supported branched linker assay system, comprising: an alphacompound and a beta compounds reversibly tethered to a solid support; abranched linker coupled to the solid support that tethers the alpha andbeta compounds to the solid support; the branched linker comprising twocleavable linkers that are chemically distinct from one another, whereina first chemically distinct linker tethers the β compound to thebranched linker and a second chemically distinct linker tethers the αcompound to the branched linker; and at least two means for cleaving thechemically distinct linkers, wherein a first cleavage means isconfigured to selectively cleave a first chemically distinct linker anda second cleavage means is configured to selectively cleave a secondchemically distinct linker.

The branched linker is a C-terminal linker system that is synthesizedonto the solid support and may comprise any number of carbons.

In embodiments, the solid support structure is two dimensional or threedimensional. Two dimensional embodiments may include but are not limitedto slides, chips, plates, or any other two dimensional solid supportstructure commonly used in the art. Three dimension solid supportstructures may include, but are not limited to molecular beads,micro-spheres, magnetic micro-spheres, test tubes, petri dishes,microcentrifuge tubes, 96 well places, or any other three dimensionalsolid support structure commonly used in the art. The solid support maybe comprised of but not limited to silicon, glass, polystyrene, latex,or any other materials commonly used as solid support structures.Further, the solid support may be comprised of any suitable twodimensional or three dimensional structure comprising a metal oxidecoating. The solid support structure may be chemically modified tofacilitate attachment of the linker system. Such modification mayinclude but are not limited to treating the surface of the supportstructure with amino silane or epoxy silane, mercaptosilanization,derivatization of the surface with aminophenyl or aminopropyl, orcoating the surface with isothiocyanate. In embodiments, the solidsupport is a polyethylene-grafted polystyrene bead.

The alpha or beta compounds utilized in the solid supported branchedlinker assay system may be small molecules, peptides, DNA/RNA aptamers,or antimicrobial peptides. In embodiments, the alpha or beta compoundsare peptoids. The alpha or beta compounds may be directly or indirectlytethered to the solid support structure. In embodiments, the alpha andbeta compounds are substantially identical.

Numerous linker systems may be appropriate for use in the presentsystem. In embodiments, the first chemically distinct linker comprises adisulfide; and the first cleavage means comprises a reducing agent. Thereducing agent employed may be any commercially available reducingagent. In embodiments, the reducing agent is dithiothreitol,mercaptoethanol, or tris-(2-carboxyethyl)phosphine. In one embodiment,the reducing agent is tris-(2-carboxyethyl)phosphine. Appropriateconcentrations of the reducing agent may be from about 1 to about 100mM. In alternate embodiments, the appropriate concentration of reducingagent may be up to about 50 mM. In still other embodiments, theconcentration of reducing agent may be up to about 25 mM. Alternatively,the concentration of reducing agent may be between about 2 to about 14mM. In one embodiment, the concentration of reducing agent is about 14mM.

In other embodiments, the second cleavable linker comprises a methionineand the second cleavage means comprises cyanogen bromide.

The solid supported branched linker assay system may further comprise ameans for screening the therapeutic effectiveness of the beta compoundand identifying the alpha compound. In non-limiting embodiments, themeans for screening comprises: a growth media that has been inoculatedwith cells of interest; the growth media further comprising the alphaand beta compounds immobilized onto the solid support and the firstcleavage means, forming a growth media complex; an incubation periodduring which the microorganism grows within and on the growth mediacomplex and the first chemically distinct linker is cleaved, therebyremoving the beta compound from the support structure; an assessmentperiod during which therapeutic effectiveness of the beta compound isassessed within the growth media complex; a removal period, wherein thesolid support and the alpha compound tethered thereto are removed fromthe growth media complex; a cleavage period wherein the second cleavagemeans selectively cleaves the second chemically distinct linker torelease the alpha compound from the support media; a means foridentifying the alpha compound; and an identification period, whereinthe alpha compound is identified.

In embodiments, the growth media utilized is soft agar. However,embodiments can be envisioned with little or no agar.

The cells of interest that are inoculated within the growth media may bemicroorganisms such as bacteria or other prokaryotic cells.Alternatively, the cells of interest may be mammalian or othereukaryotic cells.

The assessment period discussed above may comprise an analysis of theamount of cell growth inhibition that surrounds the solid support after.

In one aspect of the invention mass spectrometry is the means foridentifying at the alpha compound. Nuclear magnetic resonancespectroscopy may also be used to identify the structure of the compound.

A method is also disclosed in accordance with various embodiments of thepresent general inventive concept for identifying the effectiveness oftherapeutic compounds. The method comprises reversibly coupling an alphaand a beta compound to a solid support through a branched linker,wherein the branched linker comprises at least two cleavable linkersthat are chemically distinct from one another; the two cleavable linkersfurther comprising a first chemically distinct linker that tethers thebeta compound to the branched linker and a second chemically distinctlinker that tethers the alpha compound to the branched linker; andproviding at least two means for cleaving the chemically distinctlinkers, wherein a first cleavage means is configured to selectivelycleave a first chemically distinct linker and a second cleavage means isconfigured to selectively cleave a second chemically distinct linker.

The method additionally includes screening for the therapeuticeffectiveness of beta compound and identifying the alpha compound. Inembodiments, to accomplish the screening process, a growth media isfirst inoculated with a microorganism of interest. In some embodiments,the growth media may be agar. As stated above, alternate types of growthmedia may also be appropriate for the method.

Next, the alpha and beta compounds tethered to the solid support and thefirst cleavage means are added to the growth media, forming a growthmedia complex. The method further comprises incubating the growth mediacomplex, during which the microorganism grows within and on the growthmedia and the first chemically distinct linker is cleaved. The cleavageremoves the beta compound from the support structure. When removed fromthe support structure, the beta compound is free to interact with themicroorganism that is disposed in and around the support structurewithin the growth media complex.

The method additionally provides for assessing the therapeuticeffectiveness of the cleaved compound within the growth media complex.This assessment may be performed by analyzing the amount of cells growninhibition that surrounds the solid support, wherein a halo of inhibitedcell growth is associated with a therapeutically effective compound.This halo of inhibited cell growth can be referred to as the zone ofinhibition.

Upon finding a therapeutically effective compound, the method furtherincludes removing the solid support and alpha compound tethered theretofrom the growth media complex. Next, the embodiments of the methodinclude adding the second cleavage means to the solid support and alphacompound tethered thereto to cleave the second chemically distinctlinker. This releases the alpha compound from the support media. Thefinal step of the method may comprise identifying the therapeuticallyeffective compound. The identification may be performed by obtaining thestructure of the alpha compound. In embodiments, the structure of thecompound may be obtained through mass spectrometry. In alternateembodiments, the structure may be obtained through nuclear magneticresonance spectroscopy.

In various exemplary embodiments, the present invention comprises asolid supported branched linker system and plate based high-throughputscreening method for the identification of therapeutic compounds. Two ormore compounds are immobilized or tethered onto the solid supportthrough a branched linker that contains the two or more compounds. Thelinkers tethering the two or more compounds can be chemicallymanipulated individually. In embodiments, one compound is attached tothe linker by a disulfide, which can be cleaved with reducing reagentduring high-throughput screening to release the compound from the solidsupport to interact with the cells of interest. After identifying whichsolid supports contain effective therapeutic compound against the cellsof interest, the solid support can be removed from the screening plateand the second compound cleaved off the solid support using cyanogenbromide. This second compound can then be analyzed by mass spectrometryor nuclear magnetic resonance to identify the structure of the effectivetherapeutic compound. High-throughput screening of the compound libraryis achieved by embedding the solid supported compound library into solidagar media inoculated with the cells or microorganism of interest andcontaining a small amount of reducing reagent to cleave one linker andrelease at least one compound attached to the branched linker system. Asdescribed above, effective therapeutic compounds will result in a zoneof inhibition around the solid support, which can be removed and thesecond compound cleaved for compound identification.

In certain embodiments, the solid support used is TentaGel®, which is apolyethylene grafted polystyrene. However, as stated above, any solidsupport may be used, including but not limited to polystyrene, latexbeads, and any number of metal oxide supports. Embodiments of thebranched linker system comprise two compounds; the first may be adisulfide that can be cleaved by a reducing reagent; the second may be amethionine that can be cleaved by cyanogen bromide. Embodiments andexamples of the present invention described herein detail the use ofpeptoids immobilized on the branched linker system for antimicrobialidentification. As would be apparent to one having ordinary skill in theart, other potential therapeutic compounds could be appended to thebranched linker system in alternate embodiments, including but notlimited to small molecules, peptides, or DNA/RNA aptamers.

In several nonlimiting embodiments, the invention comprises a PeptoidLibrary Agar Diffusion (PLAD) assay, which takes advantage of asolid-phase combinatorially produced library of peptoids on a chemicallycleavable linker that can be screened within solid agar plates toreadily identify potent antimicrobial peptoids against microbes ofinterest.

Embodiments of the screening process described herein utilize solidsupported compounds embedded into lysogeny broth agar. Other potentialsolidified growth media could be used in alternate embodiments of thisscreening assay, including but not limited to Matrigel®, a gelatinousmedia that is secreted by Engelbreth-Holm-Swarm mouse sarcoma cells. Useof media such as Matrigel® is preferred in embodiments wherein the cellsof interest to be inoculated within the growth media are mammaliancells. Other suitable solid support growth matrices will be apparent tothose having skill in the art.

Other objects and advantages of this invention will become readilyapparent from the ensuing description.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows a general PLAD Assay screening schematic. (Stage 1) Solidsupport compound library is embedded into soft agar that is inoculatedwith the microorganism of interest and contains a small amount ofreducing reagent. (Stage 2) During incubation, the reducing reagentcleaves the disulfide bond, releasing the β-compound from the bead. Abacterial lawn forms with zones of inhibition forming around beads thatrelease effective antimicrobial compounds. (Stage 3) Beads exhibitingzones of inhibition are removed from the plate and the α compoundcleaved for analysis to determine the peptoid structure.

FIG. 2 shows the synthesis of branched PLAD Assay linker comprising twoparallel compounds that can be chemically manipulated individually.

FIG. 3 shows a diagram of an antimicrobial peptoid (C13_(4mer))synthesized onto the PLAD Assay linker.

FIG. 4 shows the linear MS of the complete C13_(4mer) compound.

FIG. 5 shows the linear MS of C13_(4mer) beta-strand compound.

FIG. 6 shows images from a comparative PLAD Assay screening of threereducing agents tested at five different concentrations.

FIG. 7 is a graph of the zone of inhibition/clearance as a function ofconcentration for the three reducing agents of FIG. 6.

FIG. 8 is a graph of bacterial lawn density as a function ofconcentration for the three reducing agents of FIG. 6.

FIG. 9A shows the structure of the branched linker and solid supportcomplex with two associated compounds. The α compound peptoid istethered to the solid support via a methionine linker. The β compoundpeptoid is tethered to the solid support via a disulfide linker.

FIG. 9B shows a microscopic image showing the zone of inhibitionsurrounding the PLAD linker of FIG. 9A following cleavage and release ofthe β compound.

FIG. 9C shows the linear MS of the α compound following removal fromgrown media and subsequent cleavage from the PLAD linker of FIG. 9A.

FIG. 10A shows amines incorporated into a low diversity,proof-of-concept library on the PLAD linker.

FIG. 10B shows the average zones of inhibition for the antimicrobialpeptoids identified from screening the proof-of-concept library.

FIG. 10C is a table showing the minimum inhibitory concentration (MIC)values for peptoid K15 tested against the ESKAPE pathogens.

FIG. 11 shows the chemical structure of a synthesized branched disulfidelinker attached to a polyethylene-grafted polystyrene bead.

FIG. 12 shows the chemical structure of a synthesized of PLAD linkedtest peptoid and associated polyethylene-grafted polystyrene bead.

FIG. 13 shows the amines incorporated into a low diversity,proof-of-concept library on the PLAD linker of FIG. 10A and theassociated polyethylene-grafted polystyrene bead.

FIG. 14 is a linear MS of complete test peptoid, showing the desiredcompound at 1201.2 Da (M+H) and 601.1 Da (M+2H). The disulfide bond iseasily fragmented during MS analysis, giving the complete compound minusthe β-compound at 763.7 Da (M+H).

FIG. 15 is a linear MS of the β-compound of the test peptoid at 439.4 Da(M+H), released from the PLAD linker by treatment with TCEP.

FIG. 16 is a linear MS of the α-compound of the test peptoid at 763.7 Da(M+H) after β-compound cleavage and release by TCEP.

FIG. 17 is a tandem MS of the α-compound of the test peptoid afterβ-compound cleavage and release by TCEP. The sequence of this compound(NVal-NMeo-NPhe-PLAD linker) can be confirmed using both y ions and bions.

FIG. 18 is a linear MS of the complete C13_(4mer), containing both the αand β-compounds of the peptoid.

FIG. 19 is a linear MS of the β-compound of the PLAD linked C13_(4mer),released using 1 mM TCEP.

FIG. 20 is a table showing the identity, sequence, and molecular weightof each compound in the proof-of-concept library.

FIG. 21 shows representative images from screening of the PLAD linkedproof-of-concept library. Easily visible zones of inhibition areobserved around beads releasing peptoids with varying degrees ofantimicrobial activity. Beads releasing peptoids from the library withno antimicrobial activity can be observed in the lower right image.

FIG. 22 is a linear MS of the α-compound of a peptoid hit identifiedfrom the proof-of-concept library screening. With a molecular weight of903 da, this hit was identified as sequence K15.

FIG. 23 is a tandem MS of the α-compound of a peptoid hit identified asK15 during linear MS analysis. They and b ions successfully identifiedare shown and confirm the sequence of this peptoid as K15.

FIG. 24 is a table showing the identity and sequences ofproof-of-concept peptoid library hits against E. coli ATCC 25922.

FIG. 25A shows the general structure of the proof-of-concept PLAD linkedlibrary.

FIG. 25B shows amine submonomers incorporated into each of the positionsin the library.

FIG. 25C is a homology chart from hits identified from screening of theproof-of-concept library indicating the prevalence of each submonomer ateach position.

FIG. 26 shows the structure and linear MS of K15

DETAILED DESCRIPTION OF THE INVENTION Abbreviations and Definitions

Detailed descriptions of one or more preferred embodiments are providedherein. It is to be understood, however, that the present invention maybe embodied in various forms. Therefore, specific details disclosedherein are not to be interpreted as limiting, but rather as a basis forthe claims and as a representative basis for teaching one skilled in theart to employ the present invention in any appropriate manner.

The singular forms “a,” “an,” and “the” include plural reference unlessthe context clearly dictates otherwise. The use of the word “a” or “an”when used in conjunction with the term “comprising” in the claims and/orthe specification may mean “one,” but it is also consistent with themeaning of “one or more,” “at least one,” and “one or more than one.”

Wherever any of the phrases “for example,” “such as,” “including” andthe like are used herein, the phrase “and without limitation” isunderstood to follow unless explicitly stated otherwise. Similarly “anexample,” “exemplary” and the like are understood to be nonlimiting.

The term “substantially” allows for deviations from the descriptor thatdo not negatively impact the intended purpose. Descriptive terms areunderstood to be modified by the term “substantially” even if the word“substantially” is not explicitly recited. Therefore, for example, thephrase “wherein the lever extends vertically” means “wherein the leverextends substantially vertically” so long as a precise verticalarrangement is not necessary for the lever to perform its function.

The terms “comprising” and “including” and “having” and “involving” (andsimilarly “comprises”, “includes,” “has,” and “involves”) and the likeare used interchangeably and have the same meaning. Specifically, eachof the terms is defined consistent with the common United States patentlaw definition of “comprising” and is therefore interpreted to be anopen term meaning “at least the following,” and is also interpreted notto exclude additional features, limitations, aspects, etc. Thus, forexample, “a process involving steps a, b, and c” means that the processincludes at least steps a, b and c. Wherever the terms “a” or “an” areused, “one or more” is understood, unless such interpretation isnonsensical in context.

As used herein the term “about” is used herein to mean approximately,roughly, around, or in the region of. When the term “about” is used inconjunction with a numerical range, it modifies that range by extendingthe boundaries above and below the numerical values set forth. Ingeneral, the term “about” is used herein to modify a numerical valueabove and below the stated value by a variance of 20 percent up or down(higher or lower).

Antimicrobial Peptides and Peptoids

One class of antimicrobial compounds that are not susceptible to drugresistance mechanism is antimicrobial peptides (AMPs). AMPs serve as anatural part of the host-defense innate immune system of severalorganisms. There is little known antibiotic resistance to AMPS, likelydue to their non-specific mode of killing. It is believed that most AMPscause membrane permeabilization, resulting in leakage of cytoplasmiccomponents and cell death. Other evidence indicates that some AMPs maybind to and disrupt DNA or RNA, which is not surprising given theamphipathic structure of these compounds. Although promising, AMPs havenot been developed into legitimate therapeutics due to the poorproteolytic stability and low bioavailability of peptides. Severalmimics of AMPs have been developed with the goal of preserving theiradvantages while circumventing their shortcomings. The most promising ofthese have been based on N-substituted glycines (termed peptoids) whichare similar to peptides, but with the side chain shifted from thealpha-carbon to the amide-nitrogen. Peptoids are similar to peptides infunction, yet they are not recognized by proteases and hence have aprolonged lifetime in vivo and improved bioavailability, making themexcellent candidates as therapeutics.

The development of antimicrobial peptoids has relied on the mimicry ofknown AMPs and the generation of small (<20 compound) subsets ofpeptoids. This work has generated antimicrobial peptoids that areeffective against M. tuberculosis and P. aeruginosa biofilms. The rapiddevelopment of MDR bacterial strains demands novel antibiotics and theabove mentioned efficacy of peptoids demonstrates their potential astherapeutics. The need now is to develop methods to screen very largelibraries of peptoid compounds against any bacteria of interest in arapid fashion, thereby identifying antimicrobial peptoids that can treatnew strains of MDR bacteria. Combinatorial libraries, generated bysplit-and-pool synthesis, are a way to generate large cohorts ofpotential therapeutic compounds in a relatively short period of time.These libraries are typically synthesized on the solid-phase to provideeasy manipulation during synthesis and subsequent screening.

Problems with Identification of Antimicrobial Agents

Traditionally, the greatest source of new antimicrobial agents has beenfrom natural products isolated from plants, bacteria, and otherorganisms. Though useful in the mid-1900s, identification ofantimicrobial natural products has slowed substantially due to increasedantimicrobial resistance and decreased sources of new natural products.

Detailed Description of Selected Exemplary Embodiments

In various exemplary embodiments, the invention comprises a molecularbranched linker system immobilized on a solid support which displaysparallel compounds of potential antimicrobial compounds. The linkerstethering the two parallel compounds can be chemically manipulatedindividually to allow for one compound to be released from the solidsupport during high-throughput screening, while maintaining the secondcompound on the solid support to be removed later for analysis todetermine the compound structure. Thus, one of the two identicalcompounds is utilized for potency and assessment of therapeuticassessment, while the second compound may be utilized for structuredeconvolution after screening. The invention also comprises thehigh-throughput assay, which entails embedding the solid-supportedbranch-linked potential antimicrobial compounds into agar in a Petridish inoculated with the microorganism of interest.

In several nonlimiting embodiments, the invention comprises a PeptoidLibrary Agar Diffusion (PLAD) assay, which takes advantage of asolid-phase combinatorially produced library of peptoids on a chemicallycleavable linker that can be screened within solid agar plates toreadily identify potent antimicrobial peptoids against microbes ofinterest. Embodiments of the assay rely on a unique branched system witha disulfide linker that can be chemically cleaved after embedding thelibrary into the agar. In contrast to previous bead diffusion assays,which have used photolabile linkers, the disulfide linker allows forcleavage after the beads are embedded in the agar, negating the need forUV light irradiation optimization and reducing cross contamination thatwould arise from irradiating the beads in one large batch and thenspreading them across the agar. As an additional advantage, disulfideprovides a slow-release linker, which allows for release of the tetheredcompound of interest after being embedded into a growth media, andprovides sufficient opportunity for the released compound to interactwith the microorganism within the growth media. Also, since the beads inembodiments of the present invention are surrounded by agar instead ofspread on top, the compounds have better contact with bacteria, creatingzones of clearance that are easier to read.

A central element to the PLAD Assay of the present invention is aC-terminal linker system that results in at least two identical peptoidcompounds that can be individually chemically manipulated. Inembodiments when two compounds are utilized, a first compound is termedalpha and a second compound is termed beta.

In alternate embodiments, the α and β compounds may not be identical. Insuch embodiments, the β compound may be the potential therapeutic thatis tethered to the solid support via the disulfide linker, while the αcompound is α compound that encodes for the β compound. Merely by wayexample, in embodiments, the β compound may comprise a peptoid while theα compound may comprise RNA or DNA that encodes for the β peptoid. Suchembodiments may exist in several possible iterations, wherein thecompound tethered to the disulfide linker is the therapeutic compoundand at least one other compound (termed alpha) encodes for thetherapeutic.

During the assay, soft agar may be inoculated with the microorganism ofinterest before addition of compound beads and a small amount ofreducing reagent. In embodiments, the soft agar mixture is then pouredonto a hard agar Petri dish and allowed to solidify, resulting in aneven distribution of compound beads embedded in inoculated soft agar(FIG. 1; Stage 1). Alternatively, the compounded beads may be spread onthe top of the agar mixture after solidification. The plate may then beincubated overnight, which allows for the bacteria to grow into a lawnand also results in cleavage of the disulfide bond with reducingreagent, releasing the beta-compound peptoid from the bead (FIG. 1;Stage 2). Incubation times may vary with the type cell embedded withinthe growth media. For most cell types an incubation period from 2-72hours is sufficient achieve adequate cellular growth. In someembodiments, the incubation period may be 48 hours. In otherembodiments, the incubation period may be as short as 2 hours.

When investigating antimicrobial agents, a compound that is an effectiveantimicrobial agent kills the microorganism surrounding the bead fromwhich it was released, generating an easily read zone of inhibition,wherein little or no microorganism growth is observed. A similar zone ofinhibition is observed with effective therapeutics in embodimentswherein the cell of interest are mammalian cells.

The bead embedded within the zone of inhibition can then be removed fromthe plate manually and the alpha-compound peptoid cleaved off to analyzeby mass spectrometry (MS) and MS/MS (FIG. 1; Stage 3). In embodiments,manual removal is achieved through the use of surgical tweezers.

Synthesis of the C-terminal linker for the PLAD Assay (FIG. 2) can beperformed on TentaGel® resin, a solid support which consists ofpolyethylene glycol grafted onto a polystyrene matrix. This featureallows swelling in both organic solvents, for synthesis, and aqueoussolutions, for screening. However, any commercially available solidsupport may be utilized.

The number of carbons within the branched linker that may vary. Factorsthat may influence the number of carbons in the branched linker include,but are not limited to the number of associated compounds, the type ofassociated compounds, the type of chemically distinct linkers, and thecleavage means utilized. Embodiments of the C-terminal linker comprisefrom 2-50 carbon atoms. Additional embodiments may comprise 10-30 carbonatoms, inclusive. Embodiments may comprise 15-20 carbon atoms,inclusive. The C-terminal linker may comprise 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,29, or 30 or more carbon atoms.

During synthesis in the FIG. 2 embodiment, the amino acid methionine isadded first to the resin, which provides one way to orthogonally cleavethe peptoid from the resin post-screening using cyanogen bromide,resulting in a homoserine lactone. Although methionine was utilized inthe present embodiment, other methods of orthogonally cleaving thecompound from the resin post-screening are also appropriate. One havingskill in the art would recognize that any number of solid supportchemical linkers that are commonly used in solid phase synthesis couldbe utilized in pace of methionine in alternate embodiments.

As shown in FIG. 2, after methionine addition, a spacer such asβ-alanine was added to encourage movement of the rest of the linkersystem and peptoid away from the resin. Alternative spacers including,but are not limited to other amino acids, peptoid building blocks (suchas aminohexanoic acid), or polymeric spacers (such as PEG).

In the s, the FIG. 2 embodiment, a sulfide linker can then be introducedvia peptoid submonomer methods using bromoacetic acid followed bymono-Boc protected cystamine. In alternative embodiments, amino acidsthat contain the Boc-protected disulfide linker may be incorporated intothis moiety to add the disulfide linker. In addition, the disulfidelinker could be introduced by installing a cysteine followed by anoxidation reaction with another sulfur containing compound to form thedisulfide. Additional methods of incorporating a disulfide linker willbe apparent to those having skill in the art.

After adding the disulfide linker in FIG. 2, Fmoc-aminohexanoic acid wasadded to the N-terminus of the peptomer followed by removal of the Bocand Fmoc protecting groups. Alternative amine protecting groups may alsobe utilized. These alternative groups include, but are not limited tocarboxybenzyl, tert-butyloxycarbonyl, trityl,N-1-(4,4-Dimethyl-2,6-dioxocyclohexylidene)ethyl. In this manner alinker system is produced with two free amino groups ready for peptoidsynthesis to generate identical sequences with orthogonal chemicalmanipulation. Aminohexanoic acid, or any other suitable spacer may beutilized at the branch point in order to space out the two amino groups.Alternate spacers may include but are not limited amino acids (such asβ-alanine), other peptoid building blocks, polymeric spacers (such asPEG), or small organic molecules. Without this spacer, cyclization ofthe two branches occurs during subsequent peptoid synthesis.

After completion of the linker design, an antimicrobial peptoid or otherpotentially therapeutic compound may be added. As a proof of concept, aknown antimicrobial peptoid was synthesized on the PLAD Assay linker forbacterial screening (FIG. 3). In the proof-of-concept embodiments, thepeptoid of choice, termed C13_(4mer), was designed by Barron et al., andinvolves the use of hydrophobic alkyl tails to mimic the antibacterialproperties of lipopeptides. This addition of an alkyl tail allows thepeptoid of interest to be shortened, while still retaining usefulantimicrobial behavior. One benefit of the shortening involves limitingthe number of reactions occurring in the peptoid process, allowing for ahigher yield. Another benefit is the reduction in molecular weight ofthe compound when changing from a 10-15 submonomer length peptoid to a3-5 submonomer length peptoid. The sequence of the C13_(4mer) peptoidsynthesized on the PLAD Assay linker was NTri-NLys-NPea-NPea-NLys.Nomenclature for peptoid submonomers uses standard three letter codes,as for amino acids, but prefixes the code with “N” to denote theplacement of the side chain on the amide nitrogen. Synthesis wasaccomplished by peptoid submonomer methods using bromoacetic acid andmono-Boc-1,4-diaminobutane, phenylethylamine, and 1-tridecyl amine. Theacid sensitive Boc protecting group was used on 1,4-diaminobutane, asany unprotected NLys sub-monomers would act as branching points duringbromoacylation. The Boc group was shown to still be attached aftersubsequent treatments with bromoacetic acid (data not shown)demonstrating its stability to weak acids and usefulness as a protectinggroup for this synthetic method. After final coupling of the tridecylalkyl tail, the Boc groups were removed with triflouroacetic acid (TFA)and washed thoroughly to ensure residual acid was removed. As apparentto those skilled in the art, further compounds other than peptoids, maybe tethered to the branched linker system without undue experimentation.Further compounds that may be tethered to the branched linker system ofthe present invention include, but are not limited to utilized in thesolid supported branched linker assay system may be small molecules,peptides, DNA/RNA aptamers, or antimicrobial peptides.

For proof of concept, the synthesized C13_(4mer) compound was analyzedby mass spectrometry (MS) to show the complete mass (FIG. 4) as well astested to ensure that treatment with dithiothreitol (DTT), a reducingreagent, effectively cleaved the disulfide bond, yielding the betacompound (FIG. 5). This MS analysis confirms that the present inventionenables chemical manipulation of the linkers tethering the two compoundsindividually.

Although both analyses were successful, the conditions of cyanogenbromide cleavage were optimized. In the present embodiment, conditionsof cyanogen bromide cleavage were optimized in the presence ofhydrochloric acid (HCl), however, as evident to one having skill in theart, alternate acids or concentrations of acids may be used to create asuitable environment for cyanogen bromide cleavage. In embodiments,after multiple cyanogen bromide cleavages were unsuccessful in 0.1 M HClin water, the hydrophobicity of the cleavage solution was altered withaddition of acetonitrile. After trying several different ratios, it wasdetermined that an optimal ratio of 80:20 acetonitrile:water containing0.1 M HCl resulted in the highest quality compound analysis by massspectrometry. This could be in part due to the swelling properties ofTentaGel® resin as well as a more non-polar solution helping thehydrophobic alkyl tails diffuse out of the bead and into solution. Thesedata support full enablement and optimization for synthesis, chemicalmanipulation, and mass spectroscopic analysis of the PLAD Assay linkerand the tethered compounds.

Enablement and optimization of the PLAD Assay screening conditions isdiscussed below. PLAD linked C13_(4mer) was used to evaluate the mosteffective reducing reagent for the PLAD Assay against non-pathogenic E.coli (ATCC 25922); however, the disclosed methodology is applicable to abroad array of peptoids and other compounds. Further, the cells ofinterest are not required to be a non-pathogenic E. coli. One havingskill in the art could apply similar optimization of reducing agents foruse with an array of prokaryotic or eukaryotic cells or othermicroorganisms.

Several common reducing reagents were examined at varying concentrations(0, 2, 6, 10, and 14 mM) to identify the most suitable reagent toeffectively cleave the disulfide linker without significantly affectingmicroorganismal growth. The reagents tested were dithiothreitol (DTT),β-mercaptoethanol (BME), and tris-(2-carboxyethyl)phosphine (TCEP), butany commercially available reducing reagents may be suitable for usewithin the present invention. As would be understood by one having skillin the art, the optimal reducing agent and the concentration of thereducing agent may vary with the selection of the microorganism or othercell. In any case, the effectiveness at cleaving the disulfide linkerand releasing the beta-compound peptoid may be evaluated by measuringthe zone of inhibition, which may be defined as the area around the beadwith no bacterial growth and measured from the edge of the bead to thestart of bacterial lawn growth.

In the proof-of-concept experiment, the effectiveness of reducingreagent on bacterial lawn growth was evaluated by measuring theluminosity of the light reflected by the bacterial lawn when illuminatedfrom an angle, with denser bacterial lawns resulting in greaterluminosity. All three reducing reagents resulted in concentrationdependent zones of inhibition (FIG. 6 and FIG. 7). Comparatively, TCEPprovided the clearest zones of inhibition, with the largest zones notsurprisingly observed at 14 mM. Evaluation of the bacterial lawn densityindicated that no significant effect on bacterial growth was observedfor any of the reducing reagents at the concentrations tested (FIG. 8).Given this data, TCEP at a concentration of 14 mM was used for anysubsequent PLAD Assays.

These data demonstrate the feasibility and utility of the branchedlinker system and the high-throughput PLAD Assay screening design. Thesemethods are currently being applied to identify antimicrobial peptoidsfor proof-of-concept microorganisms, but can be applied to identifyantimicrobial compounds of any sort against nearly any microorganism ofinterest.

Thus, it should be understood that the embodiments and examplesdescribed herein have been chosen and described in order to bestillustrate the principles of the invention and its practicalapplications to thereby enable one of ordinary skill in the art to bestutilize the invention in various embodiments and with variousmodifications as are suited for particular uses contemplated. Eventhough specific embodiments of this invention have been described, theyare not to be taken as exhaustive. There are several variations thatwill be apparent to those skilled in the art.

EXAMPLES

Examples are provided below to facilitate a more complete understandingof the invention. The following examples illustrate the exemplary modesof making and practicing the invention. However, the scope of theinvention is not limited to specific embodiments disclosed in theseExamples, which are for purposes of illustration only, since alternativemethods can be utilized to obtain similar results.

Example 1 Abstract

Rapid emergence of antimicrobial resistant organisms necessitatesequally rapid methods for the development of new antimicrobialcompounds. Of recent interest have been mimics of antimicrobial peptidesknown as antimicrobial peptoids, which exhibit similar potency to theformer but with improved proteolytic stability. Presented herein is ahigh-throughput method to screen libraries of antimicrobial peptoidsimmobilized on beads embedded into solid media. Termed the peptoidlibrary agar diffusion (PLAD) assay, this assay allows for individualchemical manipulation of two identical peptoid compounds. One compoundcan be released to diffuse out from a solid support bead and interactwith the microorganism during screening. The other compound can becleaved after screening from beads showing strong antimicrobial activityand analyzed by mass spectrometry to deconvolute the structure of thepeptoid. This method was applied to a small library of peptoids toidentify an antimicrobial peptoid with modest efficacy against theESKAPE pathogens.

Keywords: Peptoids, High-Throughput, Antimicrobial, CombinatorialLibrary

The increasing prevalence of multidrug-resistant (MDR) bacterialinfections in the clinic necessitates methods to rapidly identify potentnew antimicrobial agents that are effective against MDR bacteria.Antimicrobial resistance (AMR) is considered by the World HealthOrganization to be a major threat to global public health, resulting ina significant rise in global mortality rates and a significant declinein economic growth due to the growing cost of bacterial infectiontreatment.¹ A recent study predicted that by the year 2050, AMR willresult in 10 million premature deaths per year worldwide and roughly$100 trillion USD in lost economic output.² Bacterial resistance is agrowing problem due to increasing and improper use of antibioticscombined with the ability of bacteria to readily transmit informationfrom one microbe to another.^(3,4) Common mechanisms of bacterialresistance include drug efflux pumps and enzymes that break down commonantibiotics, such as β-lactamases and aminoglycosides.^(3,4) There isnow a need for antimicrobial compounds that are not susceptible to thesedrug resistance mechanisms.

One such class that has drawn particular interest lately isantimicrobial peptides (AMPs). AMPs serve as a natural part of thehost-defense innate immune system of several organisms.^(5,6) There islittle known antibiotic resistance to AMPs, likely due to theirnonspecific mode of killing.⁷ It is believed that most AMPs causemembrane permeabilization, resulting in leakage of cytoplasmiccomponents and cell death.^(5,7) Other evidence indicates that some AMPsmay bind to and disrupt DNA or RNA, which is not surprising given theamphipathic structure of these compounds.^(5,7) Although promising, AMPshave not been developed into legitimate therapeutics because of the poorproteolytic stability and low bioavailability of peptides.^(8,9) Severalmimics of AMPs have been developed with the goal of preserving theiradvantages while circumventing their shortcomings.¹⁰ One promising classof these have been based on N-substituted glycines (termed peptoids)which are similar to peptides, but with the side chain shifted from theα-carbon to the amide-nitrogen.^(9,11) Peptoids are similar to peptidesin function, yet they are not recognized by proteases and hence have aprolonged lifetime in vivo as well as improved bioavailability, makingthem excellent candidates as therapeutics.9

The development of antimicrobial peptoids has relied on the mimicry ofknown AMPs and the generation of small (<20 compound) subsets ofpeptoids.12-17 This work has generated antimicrobial peptoids that areeffective against Mycobacterium tuberculosis and Pseudomonas aeruginosabiofilms.13,14 The rapid development of MDR bacterial strains demandsnovel antibiotics, and the above-mentioned efficacy of peptoidsdemonstrates their potential as therapeutics. The need now is to developmethods to screen very large libraries of peptoid compounds against anybacteria of interest in a rapid fashion, thereby identifyingantimicrobial peptoids that can treat new strains of MDR bacteria.Combinatorial libraries, generated by split-and-pool synthesis, are away to generate large cohorts of potential therapeutic compounds in arelatively short period of time.18,19 Combinatorial libraries ofpeptoids, first synthesized by Zuckermann et al.,20 have been used toidentify inhibitors of VEGFR21 and antibody ligands,22 among otherapplications. These libraries are typically synthesized on thesolid-phase to provide easy manipulation during synthesis and subsequentscreening.18 Combined with high-throughput screening methods,combinatorial libraries represent a powerful tool for drug discovery.The work detailed here introduces a peptoid library agar diffusion(PLAD) assay, which takes advantage of a solid-phase combinatoriallyproduced library of peptoids on a chemically cleavable linker that canbe screened within solid agar plates to readily identify potentantimicrobial peptoids against microbes of interest. This PLAD assayrelies on a unique branched system with a disulfide linker that can bechemically cleaved after embedding the library into the agar. Incontrast to previous bead diffusion assays, which have used photolabilelinkers,23-25 the disulfide linker allows for cleavage after the beadsare embedded in the agar, negating the need for irradiation optimizationand reducing cross contamination that would arise from irradiating thebeads in one large batch and then spreading them across the agar. Also,since the beads are surrounded by agar instead of spread on top, thecompounds have better contact with bacteria, creating zones ofinhibition that are easier to read.

The key to the PLAD Assay is a C-terminal linker system that results intwo identical peptoid compounds, termed the alpha and beta compounds,that can be individually chemically manipulated. During the assay, softagar is inoculated with the microorganism of interest before addition ofcompound beads and a small amount of reducing reagent. The soft agarmixture is then poured onto a hard agar Petri dish and allowed tosolidify, resulting in an even distribution of compound beads embeddedin inoculated soft agar (FIG. 1, stage 1). The plate is then incubatedovernight, which allows for the bacteria to grow into a lawn and alsoresults in cleavage of the disulfide bond with reducing reagent,releasing the beta-compound peptoid from the bead (FIG. 1, stage 2). Apeptoid compound that is an effective antimicrobial agent kills themicroorganism surrounding the bead it was released from, generating aneasily read zone of inhibition. This bead can then be removed from theplate manually, and the alpha-compound peptoid cleaved to analyze bymass spectrometry (MS) and MS/MS (FIG. 1, stage 3).

Synthesis of the C-terminal linker in the PLAD Assay was done onTentaGel® resin, a solid support which consists of polyethylene glycolgrafted onto a polystyrene matrix. This feature allows swelling in bothorganic solvents, for synthesis, and aqueous solutions, for screening.The amino acid methionine is added first to the resin (FIG. 2), whichprovides a way to orthogonally cleave the peptoid from the resinpostscreening using cyanogen bromide. Resulting in a homoserine lactone,use of a methionine for orthogonal release of the compound from theresin is now common in combinatorial library synthesis andscreening.26,27 After methionine, β-alanine is added as a spacer to helpmove the rest of the linker system and peptoid away from the resin. Thedisulfide linker is then introduced via peptoid submonomer methodsl 1using bromoacetic acid followed by mono-Boc protected cystamine. Lastly,Fmoc-aminohexanoic acid was added to the N-terminus of the peptomerfollowed by removal of the Boc and Fmoc protecting groups. This producesa linker system with two free amino groups ready for peptoid synthesisto generate identical sequences with orthogonal chemical manipulation.Aminohexanoic acid was chosen to use at the branch point in order tospace out the two amino groups. Without this spacer, cyclization of thetwo branches was observed during subsequent peptoid synthesis (data notshown).

Once the initial linker design was completed, a test peptoid wassynthesized onto it to confirm the chemical manipulability of the PLADlinker. The submonomer sequence of the test peptoid was NVal-NMeo-NPhe.Nomenclature for peptoid submonomers uses standard three letter codes,as for amino acids, but prefixes the code with “N” to denote theplacement of the side chain on the amide nitrogen. Synthesis wasaccomplished by peptoid submonomer methods11 using bromoacetic acid,diisopropylcarbodiimide, and the amines isopropylamine (NVal),2-methoxyethylamine (NMeo), and benzyl amine (NPhe). The test peptoidwas analyzed by mass spectrometry (MS) to show the complete mass (FIG.14) as well as tested to ensure that treatment withtris(2-carboxyethyl)phosphine (TCEP), a reducing reagent, effectivelycleaved the disulfide bond, yielding the β-compound peptoid (FIG. 15).Lastly, the remaining α-compound peptoid after TCEP treatment wascleaved from the resin with cyanogen bromide then analyzed by MS (FIG.16) and MS/MS (FIG. 17). The resultant spectra confirmed the peptoidsequence and demonstrated the ability to deconvolute the sequence of alibrary peptoid after screening. Although all these analyses weresuccessful, the conditions of cyanogen bromide cleavage had to beoptimized. After multiple cyanogen bromide cleavages were unsuccessfulfor hydrophobic compounds in 0.1 M HCl in water, the hydrophobicity ofthe cleavage solution was altered with addition of acetonitrile. Aftertrying several different ratios (data not shown), it was determined thatan optimal ratio of 80:20 acetonitrile:water containing 0.1 M HClresulted in the highest quality compound analysis by mass spectrometry.This could be in part due to the swelling properties of TentaGel® resin,as well as a more nonpolar solution aiding diffusion of the hydrophobicpeptoid out of the bead and into solution.

With conditions optimized for synthesis, chemical manipulation, and massspectroscopic analysis of the PLAD Assay linker and peptoid compounds,we next set out to optimize PLAD Assay screening conditions. A knownantimicrobial peptoid was synthesized on the PLAD Assay linker as aproof-of-concept compound for bacterial screening (FIG. 3). The peptoidof choice, termed C134mer, was designed by Barron et al.13,14 andinvolves the use of hydrophobic alkyl tails to mimic the antibacterialproperties of lipopeptides.28 The addition of an alkyl tail allows thepeptoid of interest to be shortened, while still retaining usefulantimicrobial behavior. As shown by Barron et al.,28 incorporation of 10or 13 carbon alky tails onto pentameric antimicrobial peptoids yieldssimilar potency to peptoids that are 12 to 16 submonomers in length butwithout long alkyl tails. One benefit of the shortening involveslimiting the number of reactions occurring in the peptoid process,allowing for a higher yield. Another benefit is the reduction inmolecular weight of the compound when changing from a 10-15 submonomerlength peptoid (˜1250 Da on average) to a 3-5 submonomer length peptoid(˜400 Da on average). The sequence of the C134mer peptoid synthesized onthe PLAD Assay linker was NTri-NLys-NPea-NPea-NLys. Synthesis was againaccomplished by peptoid submonomer methods11 using bromoacetic acid,diisopropylcarbodiimide, and mono-Boc-1,4-diaminobutane (NLys),(±)-phenylethylamine (NPea), and 1-tridecylamine (NTri). The acidsensitive Boc protecting group was used on 1,4-diaminobutane, as anyunprotected NLys submonomers would act as branching points duringbromoacylation. The Boc group was shown to still be attached aftersubsequent treatments with bromoacetic acid (data not shown)demonstrating its stability to weak acids and usefulness as a protectinggroup for this synthetic method. After the final coupling of thetridecyl alkyl tail, the Boc groups were removed with trifluoroaceticacid (TFA) and the resin washed thoroughly to ensure residual acid wasremoved. The synthesized C13_(4mer) compound was analyzed by massspectrometry (MS) to show the complete mass (FIG. 18) as well as testedto ensure that treatment with TCEP effectively cleaved the disulfidebond, yielding the β-compound (FIG. 19).

PLAD linked C13_(4mer) was subsequently used to evaluate the mosteffective reducing reagent for the PLAD assay (FIG. 1) againstrelatively nonpathogenic Escherichia coli (ATCC 25922). Several commonreducing reagents were examined at varying concentrations (0, 2, 6, 10,and 14 mM) to identify the most suitable reagent to effectively cleavethe disulfide linker without significantly affecting microorganismalgrowth. The reagents tested were dithiothreitol (DTT), β-mercaptoethanol(BME), and tris(2-carboxyethyl)phosphine (TCEP). Effectiveness atcleaving the disulfide linker and releasing the beta-compound peptoidwas evaluated by measuring the zone of inhibition, defined as the areaaround the bead with no bacterial growth and measured from the edge ofthe bead to the start of bacterial lawn growth. Effect of reducingreagent on bacterial lawn growth was evaluated by measuring theluminosity of the light reflected by the bacterial lawn when illuminatedfrom an angle, with denser bacterial lawns resulting in greaterluminosity. All three reducing reagents resulted in concentrationdependent zones of inhibition (FIGS. 6 and 7). Comparatively, TCEPprovided the clearest zones of inhibition, with the largest zones notsurprisingly observed at 14 mM. Evaluation of the bacterial lawn densityindicated that no significant effect on bacterial growth was observedfor any of the reducing reagents at the concentrations tested (FIG. 8).Given this data, TCEP at a concentration of 14 mM was used for anysubsequent PLAD Assays.

To evaluate the usefulness of the PLAD Assay in identifyingantimicrobial peptoids, a very small proof-of-concept library wassynthesized on the PLAD linker using semicombinatorial chemistry (FIG.10A). Three aromatic submonomers (furfurylamine, benzylamine, and1-phenylethylamine) were randomly incorporated into the first C-terminalposition of this library, two cationic submonomers(mono-Boc-diaminoethane and mono-Boc-diaminobutane) were randomlyincorporated into the second position, and three hydrophobic submonomers(isopropylamine, 1-aminodecane, and 1-aminotridecane) were randomlyincorporated into the third position. These submonomers were chosen forthis proof-of-concept library because previous studies have shown thatpeptoids comprised of cationic and hydrophobic submonomers exhibitstrong antimicrobial activity.^(15,28,29) This produced a library with18 unique peptoid sequences (FIG. 20) that could be screened todemonstrate the utility of the PLAD Assay and identify a novelantimicrobial agent.

This library was screened against nonpathogenic E. coli. (ATCC 25922) asdescribed previously with the known C13_(4mer) antimicrobial peptoid andzones of inhibition were measured using a Leica M165FC microscope. Intotal, roughly 800 beads were screened, representing 44 replicates ofthe theoretical diversity. Multiple replicates were evaluated in onescreening to gain a better understanding of antimicrobial peptoidsequence homology and to give statistical credence to the relationshipbetween peptoid sequence and zone of inhibition. Representative imagesfrom this screening can be seen in FIG. 21. Hits, defined as beads witha measurable zone of inhibition, were isolated manually with surgicaltweezers and placed into individual tubes. These beads were boiled in 1%sodium dodecyl sulfate (SDS) to remove bacterial and media debris fromthe beads. The alpha-compound of the peptoid was cleaved from the beadusing cyanogen bromide then analyzed by MS and MS/MS to identify thestructure of the unknown peptoid. Representative spectra are given inFIGS. 22 and 23. In total 34 hits were identified (24% hit rate) and 31sequences were successfully obtained by MS and MS/MS (FIG. 24). Ahomology chart was generated to determine which residues were mostprevalent at particular positions in the identified hits (FIG. 25). Inthe first position, most antimicrobial peptoids contained benzylamine or1-phenylethylamine in equal prevalence, while very few containedfurfurylamine. There was also little difference in the abundance ofdiaminoethane and diaminobutane in the second position of identifiedhits. Interestingly, in the third position, all antimicrobial peptoidsidentified contained a 1-aminotridecane submonomer. Improvedantimicrobial activity with a long hydrophobic residue in this positionis not surprising given previously published results. The size of thezone of inhibition, presumably a measure of the peptoid's antimicrobialpotency, was also correlated with peptoid sequence (FIG. 10B). Thepeptoid with the largest average zone of inhibition was sequence K15(NTri-Nae-NPea). Note that all but one of the hits identified from thisscreening had a larger zone of inhibition than C134mer, demonstratingthe ability of even a small peptoid library in identifying potentantimicrobial agents. Interestingly, the hits with the smallest zones ofinhibition correlated to those containing furfurylamine in position 1,confirming the homology data which showed very little prevalence of thissubmonomer in identified hits.

To evaluate the efficacy of an antimicrobial peptoid identified from thePLAD Assay, the tripeptoid K15 (FIG. 26) was synthesized and its MICagainst the ESKAPE pathogens determined (FIG. 10C) The ESKAPE pathogens(Enterococcus faecium, Enterococcus faecalis, Staphylococcus aureus,Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa,and Enterobacter spp.) represent a cohort of bacteria that are resistantto most clinically used antibiotics.30 We note that the E. coli testedhere was the same strain of E. coli used during library screening (ATCC25922). K15 displayed modest efficacy against six of the seven pathogenstested with the strongest antimicrobial efficacy against A. baumanniiand E. faecium (25 μg/mL). This modest efficacy is undoubtedly due tothe limited diversity of the proof-of-concept library. However, theseresults demonstrate the capability of the PLAD Assay to identifycompounds with antimicrobial activity, even against pathogens with modesof antimicrobial resistance. Subsequent studies will focus on screeningmore diverse libraries via the PLAD Assay against each of the ESKAPEpathogens.

In summary, we have demonstrated a high-throughput screening system toidentify antimicrobial peptoids, which we believe to be modular enoughto screen any cohort of combinatorially synthesized molecules. Bydesigning a branched linker with orthogonal chemical manipulability, wehave shown that we can release the beta-compound of a peptoid using TCEPto cleave a disulfide bond during screening, producing an easily readzone of inhibition in response to effective antimicrobial peptoids,while leaving the alpha-compound still attached to the bead. MS analysisof test peptoids and a small proof-of-concept library demonstrate thefeasibility of deconvoluting the alpha-compound peptoid sequence ofstrong antimicrobial peptoids after screening, allowing researchers torapidly screen large cohorts of potential compounds without knowingtheir structure. The optimal reducing reagent conditions were determinedfor this assay and a proof-of-concept library was synthesized andscreened, subsequently identifying K15, a peptoid with modest efficacyagainst the drug resistant ESKAPE pathogens. Current efforts are focusedon screening more diverse peptoid libraries against both antimicrobialresistant bacterial and fungal pathogens.

Abbreviations

PLAD peptoid library agar diffusion

MDR multidrug resistant

AMR antimicrobial resistance

AMP antimicrobial peptide

BME β-mercaptoethanol

DTT dithiothreitol

TCEP tris(2-carboxyethyl)phosphine

REFERENCES CITED IN THIS EXAMPLE

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Antimicrobial and host-defense peptides as    new anti-infective therapeutic strategies. Nat Biotechnol. 2006;    24(12):1551-7. [PubMed]-   9. Culf A S, Ouellette R J. Solid-phase synthesis of N-substituted    glycine oligomers (alpha-peptoids) and derivatives. Molecules. 2010;    15(8):5282-335. [PubMed]-   10. Giuliani A, Rinaldi A C. Beyond natural antimicrobial peptides:    multimeric peptides and other peptidomimetic approaches. Cell Mol    Life Sci. 2011; 68(13):2255-66. [PubMed]-   11. Zuckermann R N, Kerr J M, Kent S B H, Moos W H. Efficient method    for the preparation of peptoids [oligo(N-substituted glycines)] by    submonomer solid-phase synthesis. J Am Chem Soc. 1992; 114(26):    10646-10647.-   12. Chongsiriwatana N P, Wetzler M, Barron A E. Functional synergy    between antimicrobial peptoids and peptides against Gram-negative    bacteria. Antimicrob Agents Chemother. 2011; 55(11):5399-402. [PMC    free article] [PubMed]-   13. Kapoor R, Eimerman P R, Hardy J W, Cirillo J D, Contag C H,    Barron A E. Efficacy of antimicrobial peptoids against Mycobacterium    tuberculosis. Antimicrob Agents Chemother. 2011; 55(6):3058-62. [PMC    free article] [PubMed]-   14. Kapoor R, Wadman M W, Dohm M T, Czyzewski A M, Spormann A M,    Barron A E. Antimicrobial peptoids are effective against Pseudomonas    aeruginosa biofilms. Antimicrob Agents Chemother. 2011;    55(6):3054-7. [PMC free article] [PubMed]-   15. Chongsiriwatana N P, Patch J A, Czyzewski A M, Dohm M T, Ivankin    A, Gidalevitz D, Zuckermann R N, Barron A E. Peptoids that mimic the    structure, function and mechanism of helical antimicrobial peptides.    Proc Natl Acad Sci USA. 2008; 105(8):2794-9. [PMC free article]    [PubMed]-   16. Patch J A, Barron A E. Helical peptoid mimics of magainin-2    amide. J Am Chem Soc. 2003; 125(40): 12092-3. [PubMed]-   17. Hein-Kristensen L, Knapp K M, Franzyk H, Gram L. Bacterial    membrane activity of alpha-peptide/beta-peptoid chimeras: influence    of amino acid composition and chain length on the activity against    different bacterial strains. BMC Microbiol. 2011; 11:144. [PMC free    article] [PubMed]-   18. Kennedy J P, Williams L, Bridges T M, Daniels R N, Weaver D,    Lindsley C W. Application of Combinatorial Chemistry Science on    Modern Drug Discovery. J Comb Chem. 2008; 10(3):345-354. [PubMed]-   19. Lam K S, Lebl M, Krchnak V. The “One-Bead-One-Compound”    Combinatorial Library Method. Chem Rev. 1997; 97(2):411-448.    [PubMed]-   20. Figliozzi G M, Goldsmith R, Ng S C, Banville S C, Zuckermann    R N. Methods of Enzymology. Vol. 267. Academic Press; 1996.    Synthesis of N-substituted glycine peptoid libraries; pp. 437-447.    [PubMed]-   21. Udugamasooriya D G, Dineen S P, Brekken R A, Kodadek T. A    Peptoid “Antibody Surrogate” That Antagonizes VEGF Receptor 2    Activity. J Am Chem Soc. 2008; 130(17):5744-5752. [PubMed]-   22. Gao Y, Kodadek T. Synthesis, Screening and Hit Optimization of    Stereochemically Diverse Combinatorial Libraries of Peptide Tertiary    Amides. Chem Biol. 2013; 20(3):360. [PMC free article] [PubMed]-   23. Fluxa V S, Maillard N, Page M G P, Reymond J-L. Bead diffusion    assay for discovering antimicrobial cyclic peptides. Chem Commun.    2011; 47(5):1434-1436. [PubMed]-   24. Oldenburg K R, Vo K T, Ruhland B, Schatz P J, Yuan Z. A Dual    Culture Assay for Detection of Antimicrobial Activity. J Biomol    Screening. 1996; 1(3):123-130.-   25. Silen J L, Lu A T, Solas D W, Gore M A, Maclean D, Shah N H,    Coffin J M, Bhinderwala N S, Wang Y, Tsutsui K T, Look G C, Campbell    D A, Hale R L, Navre M, DeLuca-Flaherty C R. Screening for Novel    Antimicrobials from Encoded Combinatorial Libraries by Using a    Two-Dimensional Agar Format. Antimicrob Agents Chemother. 1998;    42(6):1447-1453. [PMC free article] [PubMed]-   26. Chen X, Tan P H, Zhang Y, Pei D. On-Bead Screening of    Combinatorial Libraries: Reduction of Nonspecific Binding by    Decreasing Surface Ligand Density. J Comb Chem. 2009; 11(4):604-611.    [PMC free article] [PubMed]-   27. Kappel J, Barany G. Methionine anchoring applied to the    solid-phase synthesis of lysine-containing “head-to-tail” cyclic    peptides. Lett Pept Sci. 2003; 10(2):119-125.-   28. Chongsiriwatana N P, Miller T M, Wetzler M, Vakulenko S,    Karlsson A J, Palecek S P, Mobashery S, Barron A E. Short Alkylated    Peptoid Mimics of Antimicrobial Lipopeptides. Antimicrob Agents    Chemother. 2011; 55(1):417-420. [PMC free article] [PubMed]-   29. Mojsoska B, Zuckermann R N, Jenssen Hv. Structure-Activity    Relationship Study of Novel Peptoids That Mimic the Structure of    Antimicrobial Peptides. Antimicrob Agents Chemother. 2015;    59(7):4112-4120. [PMC free article] [PubMed]-   30. Pendleton J N, Gorman S P, Gilmore B F. Clinical relevance of    the ESKAPE pathogens. 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Example 2 Synthesis and Screening Procedures Materials and Methods

Chemicals for this project were purchased from Fisher Scientific(Waltham, Mass.), Alfa Aesar (Haverhill, Mass.), Amresco (Solon, Ohio),TCI America (Portland, Oreg.), Anaspec (Fremont, Calif.), EMD Millipore(Billerica, Mass.), Peptides International (Louisville, Ky.), andChem-Implex (Wood Dale, Ill.). Non-pathogenic E. coli (ATCC 25290) wereprovided by Dr. Mary Farone in the Department of Biology at MiddleTennessee State University (MTSU). All mass spectra were acquired oneither a Waters Synapt HDMS QToF with Ion Mobility or a ThermoScientific LTQ XL Linear Ion Trap Mass Spectrometer and all NMR spectrawere acquired on a JOEL ECA 500 NMR spectrometer. All images wereacquired using a Leica M165FC stereomicroscope and images were analyzedusing Adobe Photoshop and Microsoft Excel.

N-(tert-butoxycarbonyl)-cystamine

Cystamine dihydrochloride (4 g, 17.78 mmol) was dissolved in methanol(200 mL) and cooled to 0° C. Triethylamine (7.45 mL, 53.33 mmol) wasadded and stirred for 30 min. Boc-anhydride (4.05 mL, 17.78 mmol) wasthen added drop wise over 10 min and allowed to stir for 1 h. Thesolution was concentrated in vacuo, then washed with diethyl ether (3×30mL). 1 M NaOH solution was added to the product and extracted 2× withCH₂Cl₂. Both organic layers were combined and washed 2× with H₂O. Theorganic layer was then dried over CaCl₂ and concentrated in vacuo toyield a white solid (3.9 g, 86% yield). ESI [M+H]⁺¹ expected 253.39 Da,observed 253.1 Da. ¹H NMR (CDCl₃) δ 1.45 (s, 9H), δ 2.77 (q, 4H, J=6.19Hz), δ 3.02 (t, 4H, J=6.19 Hz), δ 3.45 (m, 2H), δ 5.02 (s, 1H).

N-(tert-butoxycarbonyl)-1,4-diaminobutane

Concentrated HCl (2.85 mL, 34.09 mmol) was added to methanol (50 mL) andcooled to 0° C. on ice. 1,4-Diaminobutane (3 g, 34.09 mmol) was added tothe mixture and stirred for 20 min. Water (ddH₂0; 7 mL) was added andstirred 30 minutes. Di-tert-butyl dicarbonate (11.72 mL, 51.11 mmol) inmethanol (30 mL) was added drop wise over 10 min then stirred for 1 h.The solvent was evaporated in vacuo and the resulting solid washed withdiethyl ether (3×30 mL). 1 M NaOH solution was added and the product wasextracted 2× with CH₂Cl₂. Both organic layers were combined and washed1× with a brine solution. The organic layer was then dried over CaCl₂and concentrated in vacuo to yield a white solid (4.81 g, 75% yield).ESI [M+H]⁺¹ expected 189.27 Da, observed 189.2 Da. ¹H NMR (500 MHz,CDCl₃): δ 4.61 (s, 1H), 3.13 (m, 2H), 2.17 (m, 2H), 1.50 (m, 6H), 1.44(s, 9H).

N-(tert-butoxycarbonyl)-1,2-diaminoethane

Concentrated HCl (3.89 mL, 46.6 mmol) was added to methanol (50 mL) andcooled to 0° C. on ice. 1,2-Diaminoethane (2.8 g, 46.6 mmol) was addedto the mixture and stirred for 20 min. Water (ddH₂0; 8 mL) was added andstirred 30 minutes. Di-tert-butyl dicarbonate (16.08 mL, 70.0 mmol) inmethanol (34 mL) was added drop wise over 10 min then stirred for 1 h.The solvent was evaporated in vacuo and the resulting solid washed withdiethyl ether (3×30 mL). 1 M NaOH solution was added and the product wasextracted 2× with CH₂Cl₂. Both organic layers were combined and washed1× with brine. The organic layer was then dried over CaCl₂ andconcentrated in vacuo to yield a white solid (2.92 g, 39.2% yield). ESI[M+H]⁺¹ expected 161.22 Da, observed 161.1 Da. ¹H NMR (500 MHz, CDCl₃):δ 4.85 (s, 1H), 3.09 (m, 2H), 2.67 (q, 2H, J=6.30 Hz), 1.46 (m, 2H),1.43 (s, 9H).

Branched Disulfide Linker Synthesis (FIG. 11)

500 mg of TentaGel® macrobeads (0.25 mmol/g loading capacity) wereswollen for 20 min in dimethylformamide (DMF). Fmoc-Met-OH (320 mg, 0.82mmol, 7 eq.) was activated withN,N,N′,N′-tetramethyl-O-(1H-benzotriazol-1-yl)uroniumhexafluorophosphate (HBTU; 330 mg, 0.82 mmol, 7 eq.) for 10 min in 10 mLDMF with 5% N-methylmorpholine (NMM; v/v). This solution was then addedto the TentaGel® resin and allowed to react for 1 h with gentle rocking.Fmoc deprotection of the methionine was accomplished using 10 mL of 20%piperidine/DMF (v/v) solution for 10 min twice. Fmoc-β-Ala-OH (270 mg,0.87 mmol, 7 eq.) was activated with HBTU (330 mg, 0.87 mmol, 7 eq.) for10 min in 10 mL DMF with 5% NMM (v/v), added to the resin and allowed toreact for 1 h with gentle rocking. Fmoc deprotection was againaccomplished with 10 mL 20% piperidine/DMF (v/v) as done before.N-(tert-butoxycarbonyl)-cystamine was next incorporated using peptoidsubmonomer synthesis.¹ Briefly, bromoacetic acid (1.38 g; 10 mmol) inanhydrous DMF (5 mL) was mixed with diisopropylcarbodiimide (DIC; 2.5mL; 16 mmol) in anhydrous DMF (5 mL) and added to the resin. Thereaction was then microwaved in a 1000 kW commercial microwave at 10%power (100 kW) for 15 s twice and rocked gently for 15 min.N-(tert-butoxycarbonyl)-cystamine (550 mg, 2.2 mmol, 17 eq.) was addedto the resin in 8 mL anhydrous DMF, microwaved at 10% power for 15seconds twice and rocked gently for 45 min. Fmoc-6-aminohexanoic acid(Fmoc-Aca-OH; 175 mg, 0.50 mmol, 4 eq.) was activated with HBTU (187 mg,0.50 mmol, 4 eq.) for 10 min in 10 mL DMF, added to the resin, andallowed to react for 1 h with gentle rocking. Boc group deprotectionfrom the cystamine side chain was then done using 10 mL of a 95%TFA/2.5% H₂O/2.5% triisopropylsilane (TIS) mixture for 1 h followed bywashing 5× with CH₂Cl₂ and 5× with DMF. Deprotection of the remainingFmoc group was done with 20% piperidine/DMF (v/v) followed by washing 4×with DMF. All reactions were tested with a ninhydrin color test, andafter each reaction the resin was washed 4× with DMF unless statedotherwise.

Synthesis of PLAD Linked Test Peptoid (FIG. 12)

Solutions of bromoacetic acid (0.417 g; 3 mmol) in anhydrous DMF (1.5mL) and DIC (0.75 mL; 4.8 mmol) in anhydrous DMF (1.5 mL) were combinedwith 50 mg of resin immobilized PLAD linker. The suspended resin wasmicrowaved twice at 10% power (100 kW) for 15 seconds, then rockedgently for 15 minutes. After the prescribed time, the mixture wasaspirated and washed four times with DMF. Benzylamine (0.643 g; 6 mmol)in anhydrous DMF (3.0 mL) was added to the resin and microwaved twice at10% power for 15 seconds followed by gentle rocking for 15 minutes. Thesuspension was aspirated and washed 4× with DMF. The previouslydescribed bromoacetic acid and DIC reaction was repeated.2-methoxyethylamine (0.451 g; 6 mmol) in anhydrous DMF (3.0 mL) wasadded, microwaved, and rocked for 15 minutes. The suspension was thenaspirated and washed 4× with DMF. Again, the bromoacetic acid and DICcoupling was repeated. Isopropylamine (0.355 g; 6 mmol) in anhydrous DMF(3.0 mL) was added to the resin, microwaved, and rocked for 15 minutes.The mixture was aspirated and washed 4× with DMF. All reactions weretested using a ninhydrin color test.

The complete test peptoid was analyzed by treating a small aliquot ofresin with 75 μL of cyanogen bromide (CNBr; 40 mg/mL) in 80:20acetonitrile (ACN):water containing 0.1 M HCl for 18 h. This solutionwas then removed in vacuo and the cleaved peptoid resuspended in 200 μLof 80:20 ACN:water containing 0.05% TFA and analyzed by MS. Theβ-compound of the test peptoid was analyzed by treating a small aliquotof resin with 500 μL of tris(2-carboxyethyl)phosphine (TCEP; 1 mM) inwater for 1 h at room temperature. The resulting supernatant was thenanalyzed by MS. The α-compound of the peptoid was analyzed by washingthe TCEP treated aliquot of resin 3× with water and subsequentlytreating with 75 μL CNBr (40 mg/mL) in 80:20 ACN:water containing 0.1 MHCl for 18 h. This solution was then removed in vacuo and the cleavedpeptoid resuspended in 200 μL of 80:20 acetonitrile:water containing0.05% TFA and analyzed by MS and MS/MS.

Synthesis of PLAD Linked C13_(4mer) (FIG. 3)

To 150 mg of resin immobilized branched disulfide linker was addedbromoacetic acid (0.414 g; 3 mmol) in anhydrous DMF (1.5 mL) and DIC(0.75 mL; 4.8 mmol) in anhydrous DMF (1.5 mL). This mixture wasmicrowaved 2× at 10% power (100 kW) for 15 seconds, rocked gently for 15minutes, and washed 4× with DMF.N-(tert-butoxycarbonyl)-1,4-diaminobutane (300 mg, 1.59 mmol) inanhydrous DMF (3 mL) was then added, microwaved 2× at 10% power for 15seconds, and reacted for 30 minutes, followed by washing 4× with DMF.The bromoacetic acid and DIC step was then repeated as described above.Phenylethylamine (2 M) in anhydrous DMF (3 mL) was added, microwaved,and allowed to react for 30 minutes, after which it was washed with 4×with DMF. These procedures were then repeated with phenylethylamine andN-(tert-butoxycarbonyl)-1,4-diaminobutane again, respectively. After anadditional bromoacetic acid/DIC coupling step, 1-aminotridecane (2 M) inanhydrous DMF (3 mL) was added to the resin, microwaved, and rockedgently for 50 minutes. Resin was then washed 4× with CH₂Cl₂ and 4× withDMF. Deprotection of remaining Boc groups was then accomplished using 8mL of a 95% TFA/2.5% H₂O/2.5% TIS solution for 1 hr. Resin was thenwashed 5× with CH₂Cl₂ and 5× with DMF. All reactions were tested using aninhydrin color test.

The complete C13_(4mer) was analyzed by treating a small aliquot ofresin with 75 μL of cyanogen bromide (CNBr; 40 mg/mL) in 80:20 ACN:watercontaining 0.1 M HCl for 18 h. This solution was then removed in vacuoand the cleaved peptoid resuspended in 200 μL of 80:20acetonitrile:water containing 0.05% TFA and analyzed by MS. Theβ-compound of the test peptoid was analyzed by treating a small aliquotof resin with 500 μL of tris(2-carboxyethyl)phosphine (TCEP; 1 mM) inwater for 1 h at room temperature. The resulting supernatant was thenanalyzed by MS.

Reducing Reagent Optimization

Solid lysogeny broth (LB) was autoclaved at 121° C. and agar plates (10mL) were poured and kept at room temperature overnight to dry them ofexcess condensation. The solid agar was plated first to serve as asupport to be overlaid with soft agar, allowing for a smooth, thin layerfor the peptoid modified TentaGel® resin to be dispersed in. Overnightculture was prepared in LB broth (5 mL) by inoculating with ATCC 25922E. coli frozen stock and incubating at 37° C. for 20 h. TentaGel® beadsfunctionalized with PLAD linked C13_(4mer) were washed 2× with H₂O thenallowed to equilibrate overnight in H₂O. Soft agar for overlay washeated to 100° C. for 30 minutes and cooled to 47° C., which kept itliquid. Compound beads were then equilibrated in 500 μL phosphate-buffersaline (PBS; pH 7.2), for each plate, for 30 minutes. Soft agar (3 mL),75 μL of E. coli overnight culture, and PLAD linked C13_(4mer) beads inPBS solution (500 μL) were then combined, and inverted 6-7 times gentlyto avoid air bubbles. This mixture, serving as the negative control withno reducing reagent, was then poured onto a hard agar plate and spreadevenly into a thin layer by manual agitation. Dithiothreitol (DTT),2-mercaptoethanol (BME), and tris(2-carboxyethyl)phosphine (TCEP) werethen tested at varying concentrations to determine effectiveness atreleasing the peptoid β-compound from the bead while maintaining goodbacterial growth. Stock solutions of 100 mmol/L DTT, BME, and TCEP wereprepared in PBS (pH 7.2). Each reducing agent was tested as describedabove by addition of the appropriate amount of stock reagent to separateindividual plates. Final concentrations of reducing reagent in the softagar overlay mixture 2, 4, 10, and 14 mmol/L for all three reducingagent. All plates were then allowed to solidify and incubated at 37° C.for 18 hours. Zones of inhibition, defined as the distance between theedge of a bead and the beginning of bacterial growth near that bead,were measured using a Leica M165FC microscope. Images were also analyzedby Adobe Photoshop to gain a measure of bacterial lawn density bymeasuring the light reflected off of the bacterial lawn when illuminatedat an angle. Both sets of analyses, zone of inhibition measurements andbacterial lawn density measurements were performed on the same plates;bacterial lawn density was measured in areas of the plate where beadswere not found.

Proof-of-Concept Library Synthesis (FIGS. 25A and 25B)

To 100 mg of resin modified with branched disulfide linker wasequilibrated in anhydrous DMF and bromoacetic acid (0.414 g; 3 mmol) inanhydrous DMF (1.5 mL) and DIC (0.75 mL; 4.8 mmol) in anhydrous DMF (1.5mL) were added. This mixture was microwaved 2× at 10% power (100 kW) for15 seconds, rocked gently for 30 minutes, and washed 4× with DMF.Anhydrous DMF (3 mL) was then added and the resin was split into threevials (1 mL each). DMF was removed from each vial and to the first wasadded 2 M furfurylamine in anhydrous DMF (2 mL), to the second 2 Mbenzylamine in anhydrous DMF (2 mL), and to the third 2 Mphenylethylamine in anhydrous DMF (2 mL). All three vials were thenmicrowaved 2× at 10% power (100 kW) for 15 seconds and rocked gently for30 minutes. The resin from the three vials was then pooled together andwashed 4× with DMF and equilibrated in anhydrous DMF. Bromoacetic/DICcoupling was then done for 30 minutes and the resin was washed 4× withDMF. Anhydrous DMF (2 mL) was added, and the resin was split into twovials (1 mL each). DMF was removed from both vials and to the first vialN-(tert-butoxycarbonyl)-1,4-diaminobutane (700 mg, 1.85 M) in anhydrousDMF (2 mL) was added, and to the second vialN-(tert-butoxycarbonyl)-1,2-diaminoethane (550 mg, 1.80 M) in anhydrousDMF (2 mL) was added. Both vials were microwaved 2× at 10% power (100kW) for 15 seconds and rocked gently for 30 minutes. The two vials werecombined and washed 4× with DMF, then equilibrated in anhydrous DMF.Bromoacetic/DIC coupling was then done for 30 minutes and the resin waswashed with DMF 4×. Anhydrous DMF (3 mL) was then added to the resin andsplit into three separate vials. The DMF was removed and to the firstvial 2 M isopropylamine in anhydrous DMF (2 mL) was added, to the secondvial 2 M 1-aminodecane in anhydrous DMF (2 mL) was added, and to thethird vial 2 M 1-aminotridecane in anhydrous DMF (2 mL) was added. Theseamine coupling vials were microwaved 2× at 10% power (100 kW) for 15seconds and rocked gently for 30 minutes. Vials were then pooled andwashed 4× with DMF and 4× with CH₂Cl₂. Deprotection of Boc groups wasaccomplished using 8 mL 95% TFA/2.5% H₂O/2.5% TIS solution for 1 hour.Resin was washed 5× with CH₂Cl₂ followed by 5× with DMF. Thissemi-combinatorial synthesis resulted in 18 unique peptoid sequencesimmobilized on the PLAD linker system for proof-of-concept testing.Ninhydrin tests were done following each successive bromoacetic acidstep and pooling of amines to show confirm successful coupling.

Proof-of-Concept Library Screening

Solid lysogeny broth (LB) was autoclaved at 121° C. and agar plates (10mL) were poured and kept at room temperature overnight to dry them ofexcess condensation. The solid agar was plated first to serve as asupport to be overlaid with soft agar, allowing for a smooth, thin layerfor the peptoid modified TentaGel® resin to be dispersed in. Overnightculture was prepared in LB broth (5 mL) by inoculating with ATCC 25922E. coli frozen stock and incubating at 37° C. for 20 h. Three aliquotsof resin (4 mg) functionalized with PLAD linked proof-of-concept librarywere washed 2× with H₂O then allowed to equilibrate overnight in H₂O.Soft agar for overlay was heated to 100° C. for 30 minutes and cooled to47° C., which kept it liquid. The resin aliquots were then equilibratedin 500 μL phosphate-buffer saline (PBS; pH 7.2) for 30 minutes. Softagar (3 mL), 75 μL of E. coli overnight culture, 500 μL of TCEP (100 mMstock; 14 mM final) and resin in PBS (500 μL) were then combined, andinverted 6-7 times gently to avoid air bubbles. This mixture was thenpoured onto a hard agar plate and spread evenly into a thin layer bymanual agitation. All plates were then allowed to solidify and incubatedat 37° C. for 18 hours. Zones of inhibition, defined as the distancebetween the edge of a bead and the beginning of bacterial growth nearthat bead, were measured using a Leica M165FC microscope. Hits, definedas beads with a measurable zone of inhibition, were isolated manuallywith surgical tweezers and placed into individual tubes. These beadswere boiled in 1% sodium dodecylsulfate (SDS) for 1 hour and washed 4×with water. The alpha compound of the peptoid was cleaved from the beadusing cyanogen bromide (50 μL; 40 mg/mL) in 80:20 acetonitrile:watercontaining 0.1 M HCl for 18 hours in the dark. This solution was thenremoved in vacuo and the cleaved peptoid resuspended in 80:20acetonitrile:water containing 0.05% TFA. MS and MS/MS analysis was thendone as previously described to identify the structure of the unknownpeptoid. In total 34 hits were identified (24% hit rate) and 31sequences were successfully obtained by MS and MS/MS.

Synthesis of K15 Peptoid

Rink Amide resin (0.101 g; 0.38 mmol/g loading) was swollen in DMF for20 minutes. After removing the DMF, 20% piperidine in DMF (5 mL) wasadded to the resin and rocked for 30 minutes. The piperidine solutionwas drained, and the beads were washed 3× with DMF. The piperidinedeprotection step was once more repeated, and the resin washed 3× withDMF. Solutions of bromoacetic acid (0.417 g; 3 mmol) in anhydrous DMF(1.5 mL) and DIC (0.75 mL; 4.8 mmol) in anhydrous DMF (1.5 mL) werecombined with the deprotected Rink Amide beads. The resin was microwavedtwice at 10% power (100 kW) for 15 seconds, then rocked gently for 15minutes; after which, the mixture was aspirated and washed four timeswith DMF. 2-phenylethylamine (0.364 g; 3 mmol) in anhydrous DMF (3.0 mL)was added to the resin and microwaved twice at 10% power for 15 secondsfollowed by gentle rocking for 30 minutes. The suspension was aspiratedand washed 4× with DMF. The previously descried bromoacetic acid and DICreaction was repeated. Boc-ethylene diamine (0.486 g; 3 mmol) inanhydrous DMF (3.0 mL) was added, microwaved, and rocked for 30 minutes.The suspension was then aspirated and washed 4× with MIT′. Again, thebromoacetic acid and DIC coupling was repeated. Tridecylamine (0.598 g;3 mmol) in anhydrous DMF (3.0 mL) was added to the resin, microwaved,and rocked for 40 minutes. The mixture was aspirated and washed 4× withDMF and 4× with CH₂Cl₂. A ninhydrin test was performed on a small sampleafter every coupling. The tripeptoid was cleaved from the resin bytreating 2× with TFA:water:TIS (95:2.5:2.5) for 1 h each. TFA wasremoved by bubbling with air and residual substance resuspended in 1:1water:acetonitrile containing 0.05% TFA. K15 was then purified byreverse phase HPLC using a C18 column and a gradient of water with 0.05%TFA to acetonitrile with 0.05% TFA. The identity of the compound wasconfirmed by MS and the solvent removed under vacuum to provide pure K15(17 mg; 17% yield).

K15 MIC Testing in ESKAPE Pathogens

Peptoid K15 was analyzed via a traditional broth minimum inhibitorconcentration (MIC) assay against seven different ESKAPE pathogens(Acinetobacter baumanii, ATCC 19606; Enterococcus faecalis, ATCC 29212;Enterococcus faecium, ATCC 19434; Escherichia coli, ATCC 25922;Klebsiella pneumoniae, ATCC 700603; Pseudomonas aeruginosa, ATCC 27853;Staphylococcus aureus, ATCC 29213). For each of the bacterial strainsscreened in the ESKAPE panel, 1-3 isolated colonies were collected froma TSA plate by a flame sanitized wire loop and resuspended in 5 mL ofTSB. The solutions were incubated at 37° C. for 18-24 hours. After thegrowth period, the turbidity was measured at 600 nm and adjusted to anoptical density of 0.08-0.13 by diluting with TSB for an approximateconcentration of 1×10⁸ CFU/mL. Once the desired OD was achieved, 20 μLof the bacteria suspension were diluted 1:20 in 380 μL Cation AdjustedMueller-Hinton broth (CAMHB) for a final concentration of 5×10⁶ CFU/mL.

4 μL of a 10 mM stock of K15 were diluted in 356 μL CAMHB for eachbacterial strain assayed (a total of 28 μL stock in 2.478 mL broth forESKAPE panel). 180 μL of this solution were delivered to three wells.For each dilution to be studied, 90 μL of the 100 μM solution werewithdrawn and delivered to 90 μL of broth This 1:2 serial dilution wascontinued to give final K15 concentrations of 100, 50, 25, 12.5, 6.3,3.1, and 1.6 μM. 90 μL of the final triplicate set being removed suchthat each well has a volume of 90 μL. A negative control containing 90μL of broth with no K15 was also prepared. 10 μL of the 1:20 dilutedbacteria were added to each well for a total volume of 100 μL. 100 μL ofbroth were delivered to a well in triplicate to serve as a mediacontrol. A tetracycline control was used, composed of 4 μL 2 mg/mLantibiotic in 356 μL broth with 40 μL bacteria. 100 μL of this solutionwere delivered to each of three wells.

The prepared plates were incubated for another 18-24 hours. Theirrespective absorbance at 600 nm was analyzed on a SpectraMax M5 PlateReader. 10 μL of PrestoBlue were added to each well and allowed toincubate for an hour. Absorbance at 555, 570, and 585 nm was analyzed todetermine viable cells having survived treatment by the antimicrobialcompound. This assay, which utilizes triplicates of each K15concentration, was ran in duplicate or triplicate for each microorganismtested on different days.

REFERENCES CITED IN THIS EXAMPLE

-   1. Zuckermann, R. N.; Kerr, J. M.; Kent, S. B. H.; Moos, W. H.,    Efficient method for the preparation of peptoids    [oligo(N-substituted glycines)] by submonomer solid-phase synthesis.    Journal of the American Chemical Society 1992, 114, (26),    10646-10647.

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain, usingno more than routine experimentation, numerous equivalents to thespecific substances and procedures described herein. Such equivalentsare considered to be within the scope of this invention, and are coveredby the following claims.

Although not to be interpreted as exclusive, specific equivalentsinclude the following. In embodiments, the pathogen or cells of interestthat are inoculated within the growth media may be prokaryotic cells,eukaryotic cells, or any organism that can grow on any type of growthmedia. For instance, in addition to the use of microorganisms discussedabove, the cells of interest may be mammalian cells. Furthermore,peptoids are only one of many compounds that may be tethered to thebranched linker system in the disclosed assay. Alternate compounds thatmay be tethered to the branched linker system of the present inventioninclude, but are not limited to utilized in the solid supported branchedlinker assay system may be small molecules, peptides, DNA/RNA aptamers,or antimicrobial peptides.

What is claimed:
 1. (canceled)
 2. (canceled)
 3. (canceled)
 4. (canceled)
 5. (canceled)
 6. (canceled)
 7. (canceled)
 8. (canceled)
 9. (canceled)
 10. (canceled)
 11. A method of identifying effective therapeutic compounds comprising: a.) reversibly coupling an alpha compound and a beta compound to a solid support through a branched linker, wherein the branched linker comprises two cleavable linkers that are chemically distinct from one another; b) the two cleavable linkers further comprising a first chemically distinct linker that tethers the beta compound to the branched linker and a second chemically distinct linker that tethers the alpha compound to the branched linker; and b.) providing at least two means for cleaving the chemically distinct linkers, wherein a first cleavage means is configured to selectively cleave the first chemically distinct linker and a second cleavage means is configured to selectively cleave the second chemically distinct linker.
 12. The method of claim 11, wherein the solid support is a polyethylene-grafted polystyrene bead.
 13. The method of claim 11, wherein the alpha or beta compounds are peptides or antimicrobial peptides.
 14. The method of claim 11, wherein the alpha or beta compounds are peptoids.
 15. The method of claim 11, wherein the alpha and beta compounds are substantially identical.
 16. The method of claim 11, wherein: a.) the first chemically distinct linker comprises a disulfide; and b.) the first cleavage means comprises a reducing reagent.
 17. The method of claim 11, wherein: a.) the second chemically distinct linker comprises a methionine; and b.) the second cleavage means comprises cyanogen bromide.
 18. The method of claim 11, further comprising screening the therapeutic effectiveness of the beta compound and assessing the identity of the alpha compound, wherein: a.) a growth media is inoculated with cells of interest; b.) adding to the growth media the alpha and beta compounds tethered to the solid support and the first cleavage means to form a growth media complex; c.) incubating the growth media complex during which the cells grow within and on the growth media and the first chemically distinct linker is cleaved, thereby removing the beta compound from the support structure; d.) assessing the therapeutic effectiveness of the cleaved compound within the growth media complex by analyzing the degree of cell growth inhibition that surrounds the solid support. e.) removing the solid support and the remaining alpha compound tethered thereto from the growth media; f.) adding the second cleavage means to the solid support and the alpha tethered thereto to cleave the second chemically distinct linker and release the alpha compound from the support media; g.) determining the identity of the alpha compound.
 19. The method of claim 18, wherein the cells of interest are microorganisms.
 20. The method of claim 18, wherein the growth media is soft agar. 